Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated (PEG), also known as poly(ethylene oxide), abbreviated (PEO), to molecules and surfaces has important applications in biotechnology and medicine. In its most common form, PEG is a linear polymer having hydroxyl groups at each terminus:HO—CH2—CH2O(CH2CH2O)nCH2CH2—OHThis formula can be represented in brief as HO-PEG-OH, where it is understood that -PEG- represents the polymer backbone without its terminal groups:-PEG- equals —CH2CH2O(CH2CH2O)nCH2CH2—PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.CH3O—(CH2CH2O)n—CH2CH2—OH.
The term poly(ethylene glycol) or PEG represents or includes all of the herein described forms of PEG and still others.
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications.HO—CH2CHRO(CH2CHRO)nCH2CHR—OHR=H and CH3 
PEG is a useful polymer having the property of water solubility as well as solubility in many organic solvents. PEG is also non-toxic and non-immunogenic. When PEG is chemically attached to a water insoluble compound, the resulting conjugate generally is water soluble as well as soluble in many organic solvents. When the molecule to which PEG is attached is biologically active, such as a drug, this activity is commonly retained after attachment of PEG and the conjugate may display altered pharmacokinetics. For example, Bentley et al. in Polymer Preprints, 38(1), 584 (1997) demonstrated that the water insoluble antemisinin becomes water soluble and exhibits increased antimalarial activity when coupled to PEG. Davis et al., in U.S. Pat. No. 4,179,337 have shown that proteins coupled to PEG have enhanced blood circulation lifetime because of reduced kidney clearance and reduced immunogenicity. The lack of toxicity of PEG and its rapid clearance from the body are advantageous for pharmaceutical applications.
As applications of PEG chemistry have become more sophisticated, there has been an increasing need for heterobifunctional PEGs, that is PEGs bearing dissimilar terminal groups:X-PEG-Ywhere X and Y are typically different functional groups. PEGs having, for example, a backbone ester group, that is to say, having an ester function interposed between polyethylene glycol polymer segments, as well as the terminal groups, X and Y:X-PEG-CO2-PEG-Yis considered to be heterobifunctional even if X and Y are the same, since each PEG segment within the overall polymer backbone is substituted in an unsymmetrical fashion.
Such heterobifunctional PEGs bearing appropriate functional groups are used to link the PEGs to surfaces or other polymers, such as polysaccharides or proteins, with the other terminus attached, for example, to a drug, a liposome, a targeting agent, a label, another protein, or a biosensor. If one terminus is bound to a polymer, and the other terminus is bonded to an appropriate functional group, cross-linking to form a hydrogel can occur.
Utilizing existing methods, however, heterobifunctional PEGs are often difficult or impossible to prepare in high purity. For example, one could conduct the reaction below, using molar equivalents of each reagent with the goal of preparing the heterobifunctional PEG acetal product shown:HO-PEG-OH+ClCH2CH(OC2H5)2+NaOH→→HO-PEG-OCH2CH(OC2H5)2+NaCl+H2OIn practice, however, some of the disubstututed PEG diethyl acetal, (C2H5O)2CH2O-PEG-OCH2CH(OC2H5)2 is also inevitably formed and some unreacted PEG also remains in the reaction mixture. Tedious chromatography is then required to separate this mixture of PEG components.
Such a chromatographic approach has been used by Zalipsky (Bioconjugate Chemistry, 4: 296-299, 1993) to purify the following heterobifunctional PEG derivative:HO-PEG-C(O)NHCH2CO2Hfrom a reaction product mixture also containing unreacted PEG and the corresponding disubstituted carboxylic acid derivative.
In certain applications, it is important that a minimal amount of HO-PEG-OH is present in the end-capped, mono-alkyl PEGs used to prepare monofunctional activated PEGs, since the presence of HO-PEG-OH often leads to doubly activated PEG derivatives which can result in crosslinked products, or have other undesirable effects. In fact, HO-PEG-OH is a common contaminant in monoalkyl PEGs such as monomethoxy-PEG. U.S. Pat. No. 5,298,410 describes a chromatographic separation of CH3O-PEG-OH from HO-PEG-OH by forming the trityl (Ph3C—) derivatives, separating the derivatives chromatographically, and then removing the trityl group from CH3O-PEG-OCPh3. International Patent Publication, WO96/35451 (Suzawa, et al.) describes benzyl PEG (C6H5—CH2—O-PEG-OH) as an intermediate in preparing a heterobifunctional PEG bearing a group at one terminus having affinity for a target cell and having a toxin at the other terminus. The benzyl PEG, however, was prepared by benzylation of PEG, followed by laborious and extensive gradient chromatography to separate benzyl PEG from dibenzyl PEG and unreacted PEG. The procedure was carried out on a small scale with a yield of only 7.8%. The method thus has little value in useful commercial production applications.
A second strategy, the polymerization approach, for preparing heterobifunctional PEGs involves anionic polymerization of ethylene oxide onto an anion, X−, which ultimately becomes the end-group of the polymer:

This method has been used by Yokoyama, et al. (Bioconjugate Chemistry, 3: 275-276, 1992) to prepare a PEG with a hydroxyl group at one terminus and an amino group at the other. Cammas, et al. (Bioconjugate Chemistry, 6: 226-230, 1995) used this method to prepare PEGs with an amino group on one terminus and a hydroxyl or methoxy group on the other. This method has also been used by Nagasaki, et al. (Biocojugate Chemistry, 6: 231-233, 1995) to prepare a PEG having a formyl group at one terminus and a hydroxyl group at the other. This method is generally useful only if X is a group suitable upon which to initiate polymerization; frequently this is not the case. Also, successful application of this method requires rigorous exclusion of water to prevent formation of HO-PEG-OH; this problem becomes more severe with increasing molecular weight of the polymer. It is also necessary to carefully control the degree of polymerization in order to obtain the desired molecular weight of the PEG derivative. This method is further limited by the potential for degradation of many types of drug molecules under the harsh conditions of the polymerization reaction if the ethylene oxide polymerization is conducted directly on the drug molecule. The method is also hampered by a lack of selectivity if more than one functional group is present on which polymerization can occur.
Thus, there is a need to provide additional and improved methods for preparing heterobifunctional PEGs that substantially eliminate at least some of the problems and drawbacks of the above-noted methods.